Gene therapy for X-Linked Adrenoleukodystrophy
X-Linked adrenoleukodystrophy (ALD) is a disease caused due to a mutation in the ABCD1 gene located on the X chromosome. ABCD1 encodes for the ALD protein, an adenosine triphosphate-binding cassette transporter located on the plasma membrane of peroxisomes of cells. Mutations in this gene results in accumulation of VLCFAs in various parts of the body, which particularly affects the adrenal glands (located above kidney), leydig cells in the testes and causes demyelination of neurons in the white matter of brain (1). Background ABCD1 gene is located at the toe end of the X chromosome at position Xq28. There are more than 600 mutations reported that cause ALD. The ALD protein encoded by ABCD1 is involved in degradation of very long chain fatty acids (VLCFAs). All fatty acids, except VLCFAs, are degraded in the mitochondria, but VLCFAs are broken down in the peroxisomes. VLCFAs include fatty acids that have more than 22 carbons. Cerotic acid (26:0) is the main type of fatty acid that accumulates in these individuals. The genotype-phenotype correlation in this disease in not very well understood. It is thought that the accumulation of VLCFAs preferably alters adrenal gland function, and in the brain tissue causes inflammation which leads to demyelination of neurons in white matter. ALD is the most common type of peroxisome disorder that affects about 1 in every 20,000 to 50,000 individuals. It has not shown to occur in any particular ethnic group. This disease is particularly in males since they have only one copy of the X chromosome, but some symptoms can also be seen in heterozygous females. There are three major types on ALD, based on the time of onset and symptoms seen: a childhood cerebral form, an adrenomyeloneuropathy type, and a form called Addison disease only. The childhood cerebral form is the most lethal and signs of the disease can be seen as early as 4 to 10 years of age. The demyelination of neurons alter their normal function of passing electric signals, resulting in behavioral problems, emotional instability, vision problems to name a few. Apart from the brain, there is impaired adrenal gland function. Symptoms and onset time, severity varies among patients. But patients usually do not survive for long after symptoms appear. Most kids with cerebral form of ALD die before adolescence (1). Diagnosis and Treatment Mass spectrophotometry-gas chromatography to detect the VLCFAs in plasma of patients is the main biochemical test, along with the genetic tests to check for mutations in the ABCD1 gene. Magnetic resonance imaging (MRI) of brain is also done to check for demyelination of white matter in the brain. If diagnosed early, allogeneic hematopoietic cell transplantation (HCT) is the only option for treating cerebral form of ALD. If not treated early, HCT would not work, and can instead cause more complications. It is usually a big challenge to obtain allogeneic stem cell donors and HCT involves a considerable risk of mortality (2). Gene therapy- A case study Due to the above mentioned drawbacks of HCT therapy for ALD, a group of scientists treated a couple of patients with HIV-1 based Lentiviral gene therapy instead of HCT as these patients did not have any matched donors or cord blood for allogeneic HCT (3, 4). The two patients, P1 and P2, had cerebral form of ALD. P1 had a large deletion from exon 6 of ABCD1, and P2 had a missense mutation of E609K in the ABCD1 gene, which results in absence of the ALD protein, as detected in fibroblasts and white blood cells using immunohistochemistry. 1.jpg|Figure 3: ALD expression in microglial cells from brain of mice that have lentiviral transduced cells. The mouse brain cells were stained with Iba-1 with green (total cells) and ALD protein with red dye. Source: reference (3). 4.jpg|Figure 4: Distribution of lentiviral vector integration sites post Hematopoietic stem cell (HSC) gene therapy for P1 and P2. (A and B) As expected lentiviral vectors integrated into chromosomes that are highly expressed. (B) Lentiviral vector integration sites in and near gene coding regions. The complete length of a gene coding region is displayed as a percentage of its complete length, with percent lentiviral vector integration sites at these positions shown for P1 © and P2 (D). TSS: Transcription start site. Source: reference (3). 5.jpg|Figure 5: Ingenuity pathway analysis (IPA) was done for the genes into which lentiviral vectors were integrated, for CD34+ hematopoietic stem cells before (dark columns) and after transplantation (light columns) from P1 (A, C) and P2 (B, D). For the pathway analysis, genes having integration sites within their gene coding regions (A, B) and genes having their integration within their gene coding regions or nearby (C, D) were analyzed. Source: reference (3). 2.jpg|Figure 6: Post hematopoietic stem cell (HSC) lentiviral gene therapy folllow up for ALD protein and very long chain fatty acid (VLCFA) levels. (A) Percentage of Peripheral Blood lymphocytes and monocytes from P1 and P2 expressing ALD protein. (B and C) Percentage of indivdual population of cells, monocytes (CD14+), granulocytes (CD15+),T lymphocytes (CD3+) and B lymphocytes (CD19+) expressing ALD protein from patients P1 (B) and P2 ©. (D) VLCFAs levels in plasma of patients expressed as a ratio of C26:0/C22:0. Source: reference (3). 3.jpg|Figure 7: Brain magentic resonance images (MRIs) from P1 (A), P2 (B) before and after gene therapy and from an untreated patient as a reference ©. Arrows point to the lesions due to ALD disease in the white matter of teh brain. P1 had lesions in fronterior lobe whereas P2 had lesions in posterior lobe. Clearly, the HSC gene therapy helped curb the damage to brain in both P1 and P2 significantly. Source: reference (3). The authors chose HIV-1 based lentiviral vectors (LV) for their advantages over other viral vectors. LVs can transduce both, dividing and non-dividing cells, have a higher transduction efficiency and allow a more efficient gene transfer than the murine gamma-retrovirus vectors, which would result together would result in higher number of cells producing the wild type ALD and having higher chance of curing the disease in the long term. LVs were designed based on defined principles for safety and to prevent formation of replication competent viruses. These LVs were used to insert a functional ABCD1 gene into the chromosome of patient cells, which would result in sustained expression and production of the functional ALD protein to compensate for absence of this protein, resulting in alleviation of the disease. Preliminary data in mice A study was first conducted in mice. Recombinant mice were created that had the ALD defect. The main drawback with these mice is that they do not show any cerabral symptoms as in humans. Nonetheless they do have defective ABCD1 gene function. ALD mouse Sca-1+ cells (functional equivalent of human CD34+ hematopoietic stem cells) were transduced with lentiviral vectors that have the wild type ABCD1 gene in vitro. These cells were transplanted back into mice and the presence of ALD protein in the brain cells monitored. ALD protein levels significantly increased in teh brain cells 12 months post transplantation (Figure 3). Procedure for Hematopoietic stem cell (HSC) gene therapy in human patients A similar strategy was used for human patients P1 and P2. Patients were given an intravenous injection of granulocyte colony-stimulating factor prior to isolation of peripher blood mononuclear cells (PBMCs). CD34+ Hematopoietic stem cells (HSC) were obtained from PBMCs by positive selection using immunomagnetic beads and pre-activated using a mixture of cytokines. The activated CD34+ cells were then infected with a replication-defective HIV-1 based lentiviral vector (CG1711 hALD) that expressed the wild type ABCD1 cDNA. The transduced cells were then cryopreserved, for infusion and test for replication competent lentiviral vectors. Transduced HSC were not designed to have a growth advantage over non-transduced cells. Therefore, P1 and P2 were given a complete myeloablation regimen using cyclophosphamide and busulfan to get rid of all the residual HSC, which would increase the chances of engraftment of transduced HSCs. The transduced HSC were then thawed and infused into P1 and P2 (4.6 x 10^6 and 7.2 x 10^6 cells per kilogram respectively). Check for possible complications The are several concerns regarding lentiviral based gene therapy. One possibility is that the LVs can produce replication competent lentiviruses (RCL) upon transduction. To rule out this, some of the infected cells CD34+ cells were co-cultured with C8166 cell line and examined for the production of RCLs. RCLs were not found in co-cultures maintained upto 3 weeks. The authors also conducted a genome wide study to check for integration sites of LV, before and after transplantation. The after transplantation analyzes included all the differentiated cells (monocytes, granulocytes, T lymphocytes, B lymphocytes among others) from a transduced HSC. LV seemed to integrate into all chromosomes of both patients (Figure 4). The integration was more in the gene coding regions, but not in any particular part of the gene coding regions (Figure 4). A pathway analysis was also conducted to check for preference of integration sites for a particular pathway, before and after transplantation (Figure 5). The LVs particularly seemed to prefer the genes that are highly expressed, i.e, genes involved in cellular groth and proliferation. Integration site anylysis for the different cell types showed that cells of myeloid and lymphoid orgin had identical inegration sites. Further analysis showed that the transduced cells did not have a clonal advantage, i.e., transduced HSC did not differentiated into any particular cell tyoe and the transduced were not present in higher numbers than usual. Results The percentage of PBMCs expressing ALD increased initially post-HSC gene therapy in both patients, followed by a decrease and then stabilization to about 10% in P1 at 30 months and 15% in P2 at 24 months (Figure 6A). A similar trent for individual cell types is also shown for P1 and P2 (Figures 6B and C). The ALD protein expression resulted in decrease of VLCFA levels, which were reduced by 20% and 28% in PBMCs of P1 and P2 respectively. As a result a decrease in overall plasma levels of VLFCAs was obtained in P1 and P2 (Figure 6D). Perhaps the main highlight of the treatment was the prevention of demyelination of white matter in teh brain cells of P1 and P2 (Figure 7). References 1. http://ghr.nlm.nih.gov/condition/x-linked-adrenoleukodystrophy 2. Shapiro, E., et al., Long-term effect of bone-marrow transplantation for childhood-onset cerebral X-linked adrenoleukodystrophy. Lancet, 2000. 356(9231): p. 713-8. http://www.ncbi.nlm.nih.gov/pubmed/11085690 PubMed. 3. Cartier, N., et al., Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science, 2009. 326(5954): p. 818-23. http://www.ncbi.nlm.nih.gov/pubmed/19892975 PubMed. 4. Cartier, N., et al., Lentiviral hematopoietic cell gene therapy for X-linked adrenoleukodystrophy. Methods Enzymol, 2012. 507: p. 187-98. http://www.ncbi.nlm.nih.gov/pubmed/22365775 PubMed.